Biosynthesized silver nanoparticles inhibit Pseudomonas syringae pv. tabaci by directly destroying bacteria and inducing plant resistance in Nicotiana benthamiana

Abstract

Silver (Ag)-containing agents or materials are widely used today in plant protection for their antimicrobial activity. In view of the superior inhibitory ability of biosynthesized (aldehyde-modified sodium alginate based) silver nanoparticles (AgNPs) against plant pathogenic fungi in our previous research, here we explored the antagonistic effect of biosynthesized AgNPs on plant pathogenic bacteria and the underlying mechanism. We selected Pseudomonas syringae pv. tabaci, the causal agent of tobacco wildfire disease, as the target and found that 1.2 μg/mL biosynthesized AgNPs completely inhibited the growth of P. syringae pv. tabaci in vitro and in vivo by partly destroying the cell membrane structure of the pathogen, resulting in cytoplasmic leakage. Moreover, Nicotiana benthamiana treated with 1.2 μg/mL biosynthesized AgNPs exhibited a significant upregulation of nonexpressor of pathogenesis-related genes 1 (NPR1) and pathogenesis-related gene 2 (PR2), the typical markers of the salicylic acid (SA)-mediated defense system, and an increase in peroxidase (POD) and polyphenol oxidase (PPO) activities as well as the production of reactive oxygen species (ROS). Furthermore, biosynthesized AgNPs treatment increased the chlorophyll content and dry weight of N. benthamiana. Overall, we demonstrated that biosynthesized AgNPs at a low concentration have high inhibitory effect on the pathogen causing tobacco wildfire disease by destroying bacterial cell membrane and inducing defense resistance in host plant. These results lay a theoretical foundation for further application of biosynthesized AgNPs in the control of plant bacterial diseases.

Background

Phytopathogenic bacteria are important plant pathogens next to plant fungi, having devastating effects on plant productivity and yield (Mansfield et al. 2012). Pseudomonas syringae, a Gram-negative bacterium, is the first among the top ten bacterial plant pathogens. So far, more than 60 pathovars have been identified in the P. syringae species complex, which can infect almost all economically important crop species and cause great losses to agriculture (Xin et al. 2018). Chemical control has been the main means for managing P. syringae, and the commonly used control agents include organic–inorganic copper pesticides and agricultural antibiotics (Gutiérrez-Barranquero et al. 2013), among which agricultural antibiotics like streptomycin and zhongshengmycin are the main agents applied in the prevention and control of plant bacterial diseases (Lyu et al. 2019; Guan et al. 2022). However, as the environment continues to deteriorate, plant bacterial diseases are occurring more frequently and causing more damage, antibiotics in large doses are thus being used to control the diseases, which makes plant bacteria increasingly resistant to antibiotics (Huang et al. 2022). As a viable alternative for the treatment of various bacteria, engineered nanoparticles have great potential to address this challenge.

Nowadays, nanomaterials are increasingly being used for disease control, as their specific physico-chemical properties allow them to more easily enter plants and interact with invasive pathogens in plants (Lee et al. 2021). Silver nanoparticles (AgNPs) are the most widely explored antibacterial nano-agent due to the broad-spectrum antimicrobial properties and robust inhibition on bacteria, viruses, and fungi (Zhang et al. 2022). Consequently, AgNPs have been used in medical device disinfection, wound treatment, and anti-infection therapy for their low resistance, diverse antibacterial mechanisms, and low dose applications (Tang and Zheng 2018). Up to now, it is generally believed that many physico-chemical properties of AgNPs such as stability, size, shape, and surface chemistry play important roles in their anti-bacterial activity (Ansari et al. 2013). Therefore, maintaining the stability and controlling the particle size of AgNPs through different strategies will help to further improve their microbial capability and broaden their biological applications.

There are various methods for synthesizing AgNPs, including flame spray pyrolysis (FSP) (Walser et al. 2011), arc plasma reactor (AP) (Cui et al. 2015), and chemical reduction (Samrot et al. 2018), with physical and chemical synthetic methods as the mainstream. While the physical and chemical routes are highly efficient, they also suffer from high cost, high by-products, low yield, and the synthesized particles are usually unstable and inhomogeneous in size. In recent years, with the development of green chemistry and nanoscience, a number of more applied and innovative synthetic strategies have been developed. Among them, the green synthesis strategies, in which nanomaterials are synthesized from natural materials, have received widespread attention due to their cost-effectiveness, environmentally friendly nature, and ease of controlling the physicochemical properties of the particles.

Our previous studies found that aldehyde-modified sodium alginate could be used in the synthesis of AgNPs, effectively enhancing the oxidation resistance of particles and reducing aggregation, and the biosynthesized AgNPs exhibited broad-spectrum resistance to plant fungi (Xiang et al. 2019). However, the antibacterial properties of the newly biosynthesized AgNPs and the underlying mechanism have not been studied. In this study, we focused on the inhibition mechanism of biosynthesized AgNPs against P. syringae pv. tabaci and their effect on resistance induction in plants. The results provide a theoretical basis for the control of plant bacterial diseases with biosynthesized AgNPs.

Results

Characteristics of biosynthesized AgNPs

The characteristic signal of Ag element was clearly observed in the energy spectrum of biosynthesized AgNPs (Fig. 1a), indicating that aldehyde-modified sodium alginate can act as a reducing agent to reduce silver ions into silver element. As shown in Fig. 1b, a specific UV absorption peak around 430 nm was observed, indicating that the scale of the synthesized particles is on the nanoscale (average diameter: 11.12 ± 3.16 nm) (Fig. 1d). Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that biosynthesized AgNPs were highly dispersed in all the observed fields (Fig. 1c, d), which confirmed that aldehyde-modified sodium alginate functions as a surfactant to improve the stability of silver particles. The above conclusion could be reconfirmed by the negative Zeta potential of biosynthesized AgNPs (normal AgNPs was positive) (Table 1). These results indicated that we obtained a nano-scale silver material with good dispersibility in water.

Fig. 1

Analysis of the characteristics of biosynthesized AgNPs. a Typical signal of Ag element in the energy spectrum of biosynthesized AgNPs. b UV–vis spectrum of biosynthesized AgNPs. c Scanning electron microscopic image of biosynthesized AgNPs. d Determination of the average particle diameter of biosynthesized AgNPs by transmission electron microscopy

Table 1 Measurement of Zeta potential

Biosynthesized AgNPs have high inhibitory activity against P. syringae pv. tabaci

We chose P. syringae pv. tabaci as the target pathogen for subsequent tests. The antibacterial properties of biosynthesized AgNPs were studied via in-dish antibacterial tests, with chloroisobromine cyanuric acid (C₃HO₃N₃ClBr) as the control agent. The results showed that biosynthesized AgNPs at different concentrations had inhibitory effects on the growth of P. syringae pv. tabaci, and the inhibitory effect was obviously concentration-dependent, with the inhibitory rate reaching 100% at 1.2 μg/mL, consistent with the inhibitory effect of the positive control C₃HO₃N₃ClBr (Fig. 2a, b). To further explore the in vivo inhibitory effect of biosynthesized AgNPs on P. syringae pv. tabaci, we sprayed 1.2 μg/mL biosynthesized AgNPs, C₃HO₃N₃ClBr (positive control), or sterilized distilled water (negative control) onto leaf surface of Nicotiana benthamiana. After incubation for 3 days, all the treated leaves were inoculated with P. syringae pv. tabaci. The results showed that the lesion sizes caused by P. syringae pv. tabaci in biosynthesized AgNPs-treated plants were visibly smaller than those in C₃HO₃N₃ClBr- or water-treated plants (Fig. 2c, d), indicating that biosynthesized AgNPs had a better in vivo antibacterial ability than the positive control C₃HO₃N₃ClBr. In general, these results demonstrate that biosynthesized AgNPs at a concentration of 1.2 μg/mL can significantly inhibit P. syringae pv. tabaci infection.

Fig. 2

Antibacterial activity of biosynthesized AgNPs in vitro and in vivo. a In vitro inhibitory effect of biosynthesized AgNPs at different concentrations against Pseudomonas syringae pv. tabaci. b Column diagram showing bacterial colony numbers on LB media containing different concentrations of biosynthesized AgNPs. c Symptoms of tobacco wildfire disease on Nicotiana benthamiana leaves pretreated with 1.2 μg/mL biosynthesized AgNPs. d Column diagram showing the size of lesions caused by P. syringae pv. tabaci in c. Mock, negative control (treated with sterilized distilled water). The data are mean ± standard error (n = 3). Different lowercase letters indicate significant difference in ANOVA univariate analysis (LSD test, P < 0.05)

Biosynthesized AgNPs inhibit the growth of P. syringae pv. tabaci by destroying its cell structure

To explore the mechanism underlying the inhibitory effects of biosynthesized AgNPs on P. syringae pv. tabaci, we cultured this bacterium in LB broth medium containing 1.2 μg/mL biosynthesized AgNPs for 5 h and then observed its cell morphology under electron microscope. As shown in Fig. 3a–d, the bacterial cells cultured in normal LB medium were plump and in the shape of cylindrical rods (Fig. 3a, c), while those cultured in LB medium supplemented with biosynthesized AgNPs were in the shape of irregularly short cylinder (Fig. 3b, d). Meanwhile, a large amount of cell lysis with cytoplasmic leakage was observed in the biosynthesized AgNPs-treated LB medium (Fig. 3b, d). These results are sufficient to support the hypothesis that biosynthesized AgNPs can destroy the normal morphology of cell membrane of P. syringae pv. tabaci. Then, we measured the growth curve of P. syringae pv. tabaci under the influence of biosynthesized AgNPs. As shown in Fig. 3e, the growth trends of P. syringae pv. tabaci in normal LB medium and in LB medium containing biosynthesized AgNPs were significantly different. The difference between bacterial growth in LB media with and without biosynthesized AgNPs reached the level of 0.001 < P < 0.01 at 3 h after incubation, and from 5 h after incubation, the difference level was kept at P < 0.001, indicating that biosynthesized AgNPs had a significant inhibitory effect on the growth of P. syringae pv. tabaci. However, the growth curve of P. syringae pv. tabaci was still in an ‘S’ shape after biosynthesized AgNPs treatment. With the increase in the concentration of biosynthesized AgNPs, the S-shaped growth curve disappeared. These results suggest that biosynthesized AgNPs inhibit the growth of P. syringae pv. tabaci by partly destroying its cell membrane structure.

Fig. 3

Biosynthesized AgNPs destroy cell structure of Pseudomonas syringae pv. tabaci. a, c Scanning electron microscopy (SEM) images showing morphological characteristics of P. syringae pv. tabaci under normal conditions. b, d SEM images showing morphological characteristics of P. syringae pv. tabaci treated with biosynthesized AgNPs (1.2 μg/mL) for 5 h. Arrows indicate disrupted bacterial cells. e Growth curve of P. syringae pv. tabaci treated with biosynthesized AgNPs. Mock, negative control (treated with sterilized distilled water). The data are mean ± standard error (n = 3). Data analysis was performed using Student’s t test (^∗0.01 < P < 0.05, ^∗∗0.001 < P < 0.01, ^∗∗∗P < 0.001)

Biosynthesized AgNPs activate antioxidant pathway in N. benthamiana

To investigate the effects of biosynthesized AgNPs on the activation of host defense, 1.2 μg/mL biosynthesized AgNPs were sprayed on N. benthamiana leaves for 3 times (once per day), with spraying sterilized distilled water as the negative control. The activities of several antioxidant-related enzymes including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), and polyphenol oxidase (PPO) were detected on the fourth day after the first spray treatment. The results showed that compared with the control group, the activities of PPO and POD in the plants treated with biosynthesized AgNPs were significantly increased (Fig. 4b, d), which were 2.24 and 1.34 times higher than those in the control group, respectively, whereas the activities of SOD and CAT were markedly decreased (Fig. 4a, c). In addition, 3,3′-diaminobenzidine (DAB) staining was used to confirm the effects of biosynthesized AgNPs on antioxidant pathway. Corresponding results showed that obvious brown spots appeared on the leaves after biosynthesized AgNPs treatment (Fig. 4e), indicating that biosynthesized AgNPs treatment could induce reactive oxygen species (ROS) production, which was consistent with the increase in POD activity in response to biosynthesized AgNPs treatment, suggesting that biosynthesized AgNPs could significantly activate plant antioxidant pathway. Taken together, biosynthesized AgNPs treatment could significantly increase the activities of POD and PPO and the level of cellular ROS in plants, thereby improving plant disease resistance.

Fig. 4

Biosynthesized AgNPs induce antioxidant activity in Nicotiana benthamiana. a–d Activities of catalase (a), polyphenol oxidase (b), superoxide dismutase (c), and peroxidase (d) were measured after biosynthesized AgNPs treatment (1.2 μg/mL) for 3 times (once per day). e DAB staining was used to detect the production of H₂O₂ in N. benthamiana after biosynthesized AgNPs treatment (1.2 μg/mL) for 3 times (once per day). Mock, negative control (treated with sterilized distilled water). The data are mean ± standard error (n = 3). Data analysis was performed using Student’s t test (^∗0.01 < P < 0.05)

Biosynthesized AgNPs induce the expression of genes involved in the salicylic acid (SA) signaling pathway

SA is one of the three major stress hormones in plants, which has long been known as a signal molecule in the induction of plant systemic acquired resistance (SAR) in plant–pathogen interaction (Ding and Ding 2020). To explore the effects of biosynthesized AgNPs on the SA signaling pathway, the expressions of two related genes, non-expressor of pathogenesis-related genes 1 (NPR1) and pathogenesis-related genes 2 (PR2), in N. benthamiana were quantified using real-time quantitative PCR after treatment with 1.2 μg/mL biosynthesized AgNPs for 3 times (once per day). As shown in Fig. 5, the expression levels of NPR1 and PR2 were significantly increased after treatment with biosynthesized AgNPs, which were 1.34 and 2.17 times higher than those in the control group, respectively, indicating that biosynthesized AgNPs could significantly activate NPR1 and PR2 to induce plant immune defense.

Fig. 5

Biosynthesized AgNPs induce the expression of salicylic acid (SA) signaling pathway-related genes NPR1 and PR2 in Nicotiana benthamiana. a Relative expression level of NPR1 after biosynthesized AgNPs treatment. b Relative expression level of PR2 after biosynthesized AgNPs treatment. N. benthamiana plants were sprayed with 1.2 μg/mL biosynthesized AgNPs for 3 times (once per day). Leaf samples were collected at next day after treatment. Mock, negative control (treated with sterilized distilled water). The data are mean ± standard error (n = 3). Data analysis was performed using Student’s t test (^∗0.01 < P < 0.05, ^∗∗∗P < 0.001)

Effects of biosynthesized AgNPs on plant growth

To investigate the effects of biosynthesized AgNPs on plant growth, we evaluated the plant height, leaf width, root length, fresh weight, dry weight, and germination rate of N. benthamiana after spraying with 1.2 μg/mL biosynthesized AgNPs for 5 times (once per day). The results showed that there were no significant differences in plant height, leaf width, root length, and germination rate between biosynthesized AgNPs-treated group and sterilized distilled water-treated group (control) (Fig. 6a–d and Table 2). Regarding dry weight and fresh weight, it was found that biosynthesized AgNPs treatment had no effect on fresh weight of plants, but significantly increased their dry weight (Table 2), indicating that biosynthesized AgNPs treatment led to an increase in the accumulation of some substances in plants. Furthermore, the chloroplast content in N. benthamiana plants treated with biosynthesized AgNPs was detected. The results showed that biosynthesized AgNPs had no significant effect on chlorophyll a content but significantly increased chlorophyll b content (Fig. 6e), indicating that biosynthesized AgNPs could significantly increase the chlorophyll content. These results all suggest that biosynthesized AgNPs are safe and have certain plant growth-promoting effects.

Fig. 6

Effects of biosynthesized AgNPs on plant growth and chlorophyll content. a–c Comparison of plant height (a), root length (b), and leaf width (c) between biosynthesized AgNPs and sterilized distilled water treated Nicotiana benthamiana plants. d Seed germination under treatment with biosynthesized AgNPs or sterilized distilled water. e Column diagram showing chlorophyll contents in plants treated with biosynthesized AgNPs or sterilized distilled water. The leaf samples were collected after treatment with 1.2 μg/mL biosynthesized AgNPs for 5 times (once per day). Mock, negative control (treated with sterilized distilled water). The data are mean ± standard error (n = 3). Data analysis was performed using Student’s t test (.^∗∗0.001 < P < 0.01)

Table 2 Effects of biosynthesized AgNPs on the growth of Nicotiana benthamiana

Discussion

Previous research has found that Ag has a significantly inhibitory effect on both Gram-negative and Gram-positive bacteria, with high biosafety but no drug resistance (Cao et al. 2022). There are several potential mechanisms of antimicrobial action of AgNPs: first, AgNPs attach to microbial cell membranes, which leads to cell wall disruption (Huang et al. 2017); second, AgNPs interact with microbial cells after penetrating into the cell, resulting in cellular dysfunctions (Xia et al. 2016; Li et al. 2017; Anuj et al. 2019); third, AgNPs produce ROS and free radical species with toxic effects after entering microbial cells (Dakal et al. 2016). Our results support the first mechanism, but we cannot rule out the possibility of the other two mechanisms, as P. syringae pv. tabaci still showed a sigmoid growth curve after treated with biosynthesized AgNPs (Fig. 3e). Lu et al. (2013) revealed that AgNPs of 15 nm completely inhibited the growth of Fusobacterium nuceatum at a concentration of 50 μg/mL, while low concentrations of AgNPs (12 and 25 μg/mL) inhibited the growth of bacteria at early stage. Nakkala et al. (2017) also reported that the synthesized AgNPs at concentrations of 60 and 100 μg/mL significantly inhibited the growth of Escherichia coli. In our experiments, we tried to use biosynthesized AgNPs at a minimum inhibitory concentration of 1.2 μg/mL in consideration of the safety that can be achieved, and found that it significantly inhibited the growth of P. syringae pv. tabaci. Although application of biosynthesized AgNPs at this concentration could not completely inhibit the growth of P. syringae pv. tabaci in vitro (Fig. 3e), it completely inhibited the growth of the pathogen in plants (Fig. 2c, d), which is the joint performance of biosynthesized AgNPs in inducing plant resistance and inhibiting bacterial growth. In general, the AgNPs we have synthesized achieve high antibacterial activity at a relatively lower dose.

Recently, metal nanomaterials have been reported to be used as immune regulator for plant disease control, a case in point is that ZnONPs can improve antiviral activity in N. benthamiana through activating resistance-related pathways, manifested by an increase in ROS production and POD and CAT activities, and upregulation of the expression of systemic resistance-related genes (Cai et al. 2019). With regard to the effects on plant antioxidant pathway, biosynthesized AgNPs treatment increased the activities of POD and PPO, but decreased the activities of CAT and SOD (Fig. 4a–d) in this study. The possible reason is that biosynthesized AgNPs entered plant cells through stomata and then bound to CAT and SOD, affecting conformations and superficial contactants of these proteins, and finally leading to the reduction of detection coefficient (Jiang et al. 2019; Liu et al. 2020b). However, we cannot rule out the possibility that the host plant inhibited the activities of CAT and SOD while eliciting POD and PPO in order to maintain the balance of oxidative pathways in vivo. SA plays a key signaling role in activating plant defense responses following pathogen attack (Klessig et al. 2000). NPR1 is a protein with typical WD40-binding domain and localized in nucleus. As a key regulator in the signal transduction pathway that leads to SAR, NPR1 can regulate the activation of R gene and trigger plant resistance against pathogens in the SA signaling pathway (Kumar et al. 2022). Pathogenesis-related proteins, known as PR proteins, are closely related to plant disease resistance (Bol et al. 1990). In response to pathogen challenge, plants usually up-regulate PR protein expression to enhance defense (Cutt et al. 1989). PR proteins are generally formed at pathogen infection sites, and different PR proteins have different functions. PR2 has β-1, 3-glucanase activity, catalyzing the hydrolysis of glucan into oligosaccharides that can be used as signal molecules to activate host defense response (Lv et al. 2020). In this study, we found that biosynthesized AgNPs could significantly increase the expression of NPR1 and PR2 in N. benthamiana (Fig. 5), suggesting that biosynthesized AgNPs could induce the SA signaling pathway, thus allowing plants to acquire systemic resistance.

Biocompatibility studies have shown that silver particles at low concentrations have no obvious toxic and side effects on cells of fish and human, but cause serious toxicity at high concentrations (Munger et al. 2014; Mohamed et al. 2021). Previous studies have confirmed that silver particles can effectively inhibit a variety of plant bacteria, but their safety in plants has not been reported yet. In this study, no significant phenotypic differences were observed in plants treated with or without biosynthesized AgNPs at a concentration of 1.2 μg/mL, except for the significant increase in dry weight and chlorophyll content (Fig. 6 and Table 2). These results further confirmed that biosynthesized AgNPs have excellent biocompatibility with promising applications in plant disease control.

Taken together, in this study, we developed a simplified and efficient biosynthesized AgNPs synthesis protocol, namely, synthesis of AgNPs using aldehyde-modified sodium alginate. Our results demonstrated that biosynthesized AgNPs had high inhibitory effect on plant pathogenic bacteria at low concentrations. We confirmed that biosynthesized AgNPs mainly exerted their antibacterial activity through inhibiting bacterial growth, destroying cell membranes of bacteria, activating the SA signaling pathway, and inducing the activities of disease-resistance related enzymes in host plants. More importantly, the study confirmed that biosynthesized AgNPs with bidirectional antibacterial mechanism had no negative effect on the basic growth indexes of plants.

Conclusions

In this study, biosynthesized AgNPs were synthesized, which have very strong inhibitory effect on P. syringae pv. tabaci at a low concentration. By destroying the cell structure of bacterial pathogen and inducing resistance in host plant, the dual inhibitory effects on plant pathogenic bacteria were achieved. The results lay a theoretical basis for the application of biomodified silver in crop production in the future.

Methods

Materials and instruments

Tobacco (N. benthamiana) plants were cultured in a growth chamber under a photoperiod of 14-h light/10-h dark at 25 °C and 75% humidity. Fresh leaves were collected and frozen in liquid nitrogen for subsequent RNA isolation. Sodium alginate (viscosity 200 ± 20 mPa.s) (Aladdin, Shanghai, China), C₃HO₃N₃ClBr (Henan Yintian Fine Chemical, Henan, China), transmission electron microscope (JEM-1200EX, JEOL, Japan), scanning electron microscope (su8020, Hitachi, Japan), Zetasizer Ultra (Malvern ZEN3600, UK), and ultraclean workbench (AIRTECH, Jiangsu, China) were used in this study.

Synthesis of biosynthesized AgNPs

Here, the synthesis scheme of AgNPs (Xiang et al. 2019) we developed previously was modified. The simplified protocol is as follows: 0.4 g aldehyde-modified sodium alginate and 0.2 g silver nitrate were dissolved in 100 mL deionized water; 5 mL 2% ammonia solution was added (yellow flocs were observed to disappear); the mixed solution was heated at 70 °C for 7 min to obtain biosynthesized AgNPs. The final biosynthesized AgNPs were purified via centrifugation (8000 g, 5 min, 4 °C, repeated 3 times) and then dialyzed for 5 days.

Characterization of biosynthesized AgNPs

SEM, Zeta potential analysis, TEM, and Spectrophotometry were used to determine the formation of silver particles and their basic structure. Briefly, SEM and TEM were used to observe the morphology and dispersion of nanoparticles. Spectrophotometry was used to detect the absorption signal of biosynthesized AgNPs (different particle sizes have different absorption locations). Finally, Zeta potential analysis was used to detect the surface potential of biosynthesized AgNPs.

Optimal concentration screening of biosynthesized AgNPs

P. syringae pv. tabaci was selected for antimicrobial studies, and 50 μL of bacterial suspension (1.0 × 10⁹ CFU/mL) was evenly spread over the solid LB medium containing different concentrations of biosynthesized AgNPs (0.075, 0.15, 0.3, 0.6, and 1.2 μg/mL), with C₃HO₃N₃ClBr (diluted 1000-fold) and sterilized distilled water as positive and negative controls (Jiang et al. 2021), respectively. After incubation at 25 °C for 48 h, the colony growth was evaluated and the inhibitory effects of biosynthesized AgNPs on P. syringae pv. tabaci in different treatments were determined.

Determination of growth curve

Growth curve reflects the vitality of an organism in a specific environment (Karahan et al. 2016). To determine the effect of biosynthesized AgNPs on the growth curve of P. syringae pv. tabaci, 30 μL of P. syringae pv. tabaci suspension (1.0 × 10⁹ CFU/mL) was added to 30 mL liquid LB medium containing biosynthesized AgNPs at a concentration of 1.2 μg/mL. In control group, biosynthesized AgNPs was replaced by sterilized distilled water. The bacterial suspension was cultured at 30 °C with an agitation speed of 200 rpm. The absorbance of cell suspension at 600 nm was determined once an hour for 24 h with an UV–visible spectrophotometer. All treatments were repeated three times.

Determination of the interaction between biosynthesized AgNPs and P. syringae pv. tabaci

SEM was used to observe cell morphology of P. syringae pv. tabaci. The bacterial suspension (200 μL, OD₆₀₀ = 1.0) was added into 40 mL LB broth medium with or without biosynthesized AgNPs (a final concentration of 1.2 μg/mL). After incubation on a shaker with an agitation speed of 220 rpm at 28 °C for 5 h, the cultures were centrifuged at 3500 g for 15 min, and the obtained bacterial cells were fixed in 2.5% glutaraldehyde for 2 h. The fixed bacteria were washed twice with phosphate buffer (PBS) to remove the glutaraldehyde residues and then were dehydrated through a graded series of ethanol (30, 40, 50, 60, 70, 80, 90, and 100%). After that, the dehydrated bacteria were subjected to critical point drying and gold coating prior to observation under a scanning electron microscope.

Enzyme activity determination

The enzyme activity was determined according to the previous report (Lv et al. 2020). PBS was used to extract total protein from the treated N. benthamiana leaves, and enzyme activity kits were used to measure the activities of PPO (at 410 nm), POD (at 470 nm), SOD (at 560 nm), and CAT (at 240 nm) (Sinobest, YX-C-A404, YX-C-A502, YX-C-A500, YX-C-A501) according to the manuals, with three repetitions for each treatment.

DAB staining

DAB staining is commonly used to detect peroxidase activity in cells (Liu et al. 2020a). With spraying sterilized distilled water as control, 1.2 μg/mL biosynthesized AgNPs were sprayed on N. benthamiana leaves. After spraying for 3 times (once per day), the leaves were placed in DAB staining solution and incubated in the dark at room temperature on a shaker with a shaking speed of 50–100 rpm for 4 h. The staining solution was prepared as follows: 50 mg DAB (Sangon, A690009) was dissolved in a centrifuge tube with 45 mL deionized water, then the pH of the solution was adjusted to 3.0 with HCl, and finally 25 μL Tween-20 and 2.5 mL 200 nM Na₂HPO₄ (the solution was freshly prepared just before use) were added. After dyeing, decolorizing solution (ethanol: acetic acid: glycerol = 3:1:1) was added to decolorize the leaves in 95 °C water bath for 15 min.

Antimicrobial activity test

With spraying sterilized distilled water as the negative control and C₃HO₃N₃ClBr as the positive control, 1.2 μg/mL biosynthesized AgNPs were sprayed on N. benthamiana leaves. After spraying for 3 times (once per day), 20 μL of P. syringae pv. tabaci suspension (1.0 × 10⁹ CFU/mL) was dropped into the back of the leaves via the pinholes punctured with a needle, and the inoculated leaves were covered with a filter paper whose surface was dropped with another 20 μL of P. syringae pv. tabaci suspension (1.0 × 10⁹ CFU/mL) to keep moisture. After that, the leaves were incubated at 28 °C for 5 days (Cai et al. 2021).

Real-time quantitative PCR detection

Leaf samples were collected after spraying with biosynthesized AgNPs for 3 times (once per day) and kept at −80 °C. The RNA was extracted by Eastep Super Total RNA Extraction Kit (Promega). First strand cDNA was obtained from reverse-transcription of total RNA (1 μg) using the PrimeScript RT reagent kit (TaKaRa). QTOWER2.0 real-time PCR (Analytikjena, Germany) and SYBR Prime qPCR Set (BioGround, BG0014) were used to analyze the relative expression levels of target genes. Primer3web (https://bioinfo.ut.ee/primer3/) was used to design gene-specific primers based on the coding-sequence of each gene. NbACTIN was selected as an internal reference, and the relative changes of gene transcription level were quantified by the 2^–△△Ct method (Liu et al. 2022). The primers were listed in Table 3.

Table 3 Primers used in this study

Determination of chlorophyll content

After surface sterilization with 75% ethanol, the sampled leaves were cut into small pieces, 1 g of which were randomly weighed and soaked in 100% acetone solution in the dark for 12 h until the leaves were completely degreased. Finally, the contents of chlorophyll a (at absorption wavelength of 661.2 nm) and chlorophyll b (at absorption wavelength of 644.8 nm) in the soaking solution were determined by an UV–visible spectrophotometer, and the total chlorophyll content was calculated according to the following formula: Total chlorophyll = chlorophyll a + chlorophyll b.

Effects of biosynthesized AgNPs on plant growth

In the seed germination test, N. benthamiana seeds were disinfected in 2.5% sodium hypochlorite solution for 15 min and then washed with sterilized distilled water for three times until the sodium hypochlorite residue was completely removed. Biosynthesized AgNPs were diluted in equal ratio with sterilized distilled water to a concentration of 1.2 μg/mL, and the same volume of sterilized distilled water was used as control. After drying, the seeds were transferred to petri dishes containing filter papers which were wetted by biosynthesized AgNPs or sterilized distilled water, and 40 seeds were placed in each dish and spread evenly. These seeds were exposed to light for 14 h with a relative humidity 85% at 25 ± 2 °C. Each process was repeated three times. In the growth test, the plants were sprayed with 1.2 μg/mL biosynthesized AgNPs for 5 times (once per day), with spraying sterilized distilled water as the control. Then the plants were placed in the greenhouse. After 20 days, the plant height, root length, leaf width, dry weight, and fresh weight were measured.

Data analysis

All experiments in this study were performed with at least 3 replicates, and the data are represented as the mean ± standard error (SE, n = 3). Statistical analysis was performed using SPSS Student’s t test (^*0.01 < P < 0.05, ^**0.001 < P < 0.01, ^***P < 0.001) and ANOVA analysis (LSD test, P < 0.05).